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Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin
Postharvest Biology and Technology 25 (2002) 209– 220 www.elsevier.com/locate/postharvbio
Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin David A. Brummell *, William J. Howie, Christa Ma, Pamela Dunsmuir DNA Plant Technology, 6701 San Pablo A6enue, Oakland, CA 94608, USA Received 25 July 2001; accepted 6 October 2001
Abstract Expansins are proteins that cause cell wall loosening, and are involved in many aspects of cell wall modification during development. In tomato, the expansin gene LeExp1 shows ripening-related accumulation of mRNA and protein, and transgenic silencing of the expression of this gene results in tomato fruit that are significantly firmer than corresponding azygous controls throughout ripening. Examination of postharvest quality characteristics of fruit suppressed in accumulation of LeExp1 protein found that increased firmness resulted in significantly improved fruit integrity during storage at 13 °C. Based upon the first appearance of noticeable deterioration, fruit shelf life was extended by 5–10 days, depending upon the packaging. However, the increased firmness of LeExp1-suppressed fruit did not result in increased resistance to the necrophytic pathogens Botrytis cinerea and Alternaria alternata. Juice prepared from LeExp1-suppressed fruit following a microwave break had a soluble solids content (°Brix), insoluble solids content (precipitate weight ratio) and serum viscosity similar to controls. Resuspension of the insoluble pelleted particulate material in 15% of the serum produced a thick paste, which allowed estimation of gross viscosity in a Bostwick consistometer. The viscosity of paste from LeExp1-suppressed fruit was 19% greater than that from corresponding azygous controls, presumably due to changes in the insoluble particulate components affecting flow characteristics. No significant effects of the LeExp1 transgene on fruit size or fruit number per plant were noted. The data suggest that fruit suppressed in expression of LeExp1 have improved shelf life and processing properties. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Expansin; Fruit quality; Processing; Ripening; Shelf life; Tomato
1. Introduction Fruit quality has many aspects, including not only the obvious properties of flavor, color, nutri* Corresponding author. Present address: Department of Pomology, University of California, Davis, CA 95616, USA. Tel.: +1-530-752-3971; fax: + 1-530-752-8502. E-mail address: [email protected] (D.A. Brummell).
tional content and firmness, but also shelf life, processing qualities and resistance to pre- and postharvest pathogens. Excessive softening is the main factor responsible for the deterioration that limits shipping, storage and saleability, and fruit firmness and texture also affect the integrity of chopped and diced fruit used for canning and fruit products. In the case of tomato, firmness and texture influence the viscosity of processed juice
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and paste, which is a major component of value (Thakur et al., 1996c). Reducing fruit softening during ripening enhances shelf life and reduces spoilage and waste, and is of great commercial interest. Fruit softening during ripening is brought about by a disassembly of the fruit cell wall, the major changes including depolymerization and solubilization of pectins, depolymerization of matrix glycans (hemicelluloses), and loss of pectic galactose side chains (Brummell and Harpster, 2001). The cell wall-modifying proteins that bring about changes in cell wall structure and its component polysaccharides have not all been identified, but transgenic suppression of ripening-related enzymes involved in pectin metabolism resulted in improvements in various aspects of quality. Suppression of polygalacturonase (PG) activity caused only a very small reduction in fruit softening with ripening, but extended fruit shelf life, reduced fruit susceptibility to postharvest pathogens and increased the viscosity of juice and paste prepared from these fruit (Schuch et al., 1991; Kramer et al., 1992; Langley et al., 1994). Similarly, transgenic suppression of pectin methylesterase (PME) activity had little effect on fruit softening during ripening, but processed juice and paste showed higher soluble solids content and increased viscosity (Tieman and Handa, 1994; Tieman et al., 1992, 1995; Thakur et al., 1996a). Thus, increased fruit quality can result from reduced cell wall breakdown during ripening, not necessarily an improvement in firmness but an improvement in fruit integrity and in the properties of processed fruit products. The matrix glycan components of the cell wall also become depolymerized during ripening (Tong and Gross, 1988; Maclachlan and Brady, 1994), and this is a major contributor to fruit softening. Enzymes which might be involved in the breakdown of matrix glycans include endo-1,4-b-glucanases (EGases), xyloglucan endotransglycosylases and expansins (Brummell and Harpster, 2001). Expansins are cell wall-localized proteins that possess a structure with similarities to carbohydrate-binding proteins (Shcherban et al., 1995), and are believed to act in the wall by disrupting the non-covalent binding between matrix glycans
and cellulose microfibrils (Cosgrove, 2000). They are encoded by large gene families, consisting of at least 24 genes in Arabidopsis (Cosgrove, 2000). Expansins bring about the cell wall loosening responsible for growth (McQueen-Mason et al., 1992), and several expansin gene family members are expressed in expanding and maturing green tomato fruit (Brummell et al., 1999a; Catala et al., 2000). Expansins are also involved in other developmental processes such as abscission (Cho and Cosgrove, 2000), and in tomato the mRNA of one expansin gene, LeExp1, was found to accumulate specifically during fruit ripening and showed the regulation by ethylene characteristic of ripening-related genes in climacteric species (Rose et al., 1997). Suppression of LeExp1 mRNA and protein accumulation in transgenic fruit reduced softening during ripening, whereas overexpression of LeExp1 mRNA and protein increased fruit softening (Brummell et al., 1999b). Modification of LeExp1 protein levels in fruit was found to result in changes in cell wall polymer metabolism, and it appears LeExp1 is an important component of the cell wall-modifying machinery. Since suppression of the ripening-related expansin LeExp1 reduced fruit softening during ripening, the aim of this work was to examine if improved fruit firmness also resulted in enhancements to other aspects of fruit quality, such as fruit shelf life, disease resistance and the viscosity of processed products. Two transgenic lines were selected from two large populations of primary transformants possessing LeExp1 transgenes under the control of the constitutive cauliflower mosaic virus 35S promoter (Brummell et al., 1999b). Primary transformant 17-42 was transformed with a complete, functional LeExp1 open reading frame, whereas line 18-40 was transformed with a truncated version that could not produce functional protein from the transgene. These two lines showed strong silencing of both the endogenous LeExp1 gene and the transgene, and in red ripe fruit homozygous progeny possessed approximately 5 and 3%, respectively, of wild type levels of immunodetectable LeExp1 protein (Brummell et al., 1999b). Seeds from these lines homozygous for the transgene were the transgenic material used in this study.
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2. Materials and methods
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(including previously-harvested and dropped fruit) was used as an estimate of fruit set.
2.1. Plant material 2.3. Shelf life Tomato (Lycopersicon esculentum Mill., cv ‘Ailsa Craig’) plants suppressed in the expression of the ripening-related expansin gene LeExp1 were generated by constitutive expression of sense LeExp1 transgenes as previously described (Brummell et al., 1999b). Primary transformants (T1 generation) 17-42 and 18-40 showing strong suppression of LeExp1 mRNA and protein accumulation were allowed to self-fertilize, and seeds were collected from homozygous T2 generation plants 17-42-5 and 18-40-11, and from corresponding azygous sibling plants. Two populations of T3 generation plants (each consisting of 16 plants of each genotype and 16 wild type controls) were planted in September 1998 and February 1999, respectively, and were grown to maturity in a greenhouse. The first population was used for studies of yield characteristics and disease resistance, and the second for evaluation of shelf life. A third population of 40 plants each of genotypes 18-40-11 and 18-40-azygous for a larger-scale repeat of shelf life properties and for fruit juice and paste production was planted in May 1999. Thus for each line (either 17-42 or 18-40) there was a homozygous transgenic population (progeny of 17-42-5 or 18-40-11) and an azygous control population that was no longer transgenic, since the transgene had been eliminated by segregation, but the plants of which were derived from the same cell line during tissue culture.
Fruit were harvested at breaker (the first appearance of red color on the exterior of the fruit), turning (up to 10% red) or early pink (10–30% red) ripening stages, and surface washed in large volumes of water containing 2.5 ml commercial bleach per liter, then rinsed with tap water. Fruit were blotted then air dried and sorted, and fruit with any abnormalities or damage were discarded. For packing in boxes, fruit were packed at this ripening stage. Preliminary experiments consisted of three replicates each of eight fruit per genotype, packed in random blocks in flat cardboard shipping boxes containing plastic liners holding 25 (5× 5) fruit. Boxes were stored at 13 °C. For packing in plastic clam shells, fruit were ripened at room temperature until red ripe (approximately 7 days after harvest) before packing. A preliminary experiment consisted of three clam shells filled with fruit (eight to 12 fruit, depending on fruit size) per genotype. Clam shells were stored at 13 °C. Fruit were assessed for deterioration on a five-point scale (see Section 3) at intervals. Subsequent experiments used the 18-40-11 and 18-40-azygous genotypes only. For boxes, six replicates each consisting of eight fruit were used per genotype. For clam shells, three replicates each consisting of eight or nine fruit were used per genotype. Fruit were assessed for deterioration on a five-point scale twice per week.
2.2. Agronomic properties
2.4. Disease resistance
For measurements of fruit size, initial experiments found that fruit diameter and fruit weight were directly correlated (data not shown), suggesting that none of the genotypes had an effect on fruit shape. Thereafter, only fruit weight was recorded, and data shown are means based on measurements from a minimum of 150 fruit per genotype. All fruit harvested from each plant were recorded. When plants were mature and all flowers had set fruit, remaining fruit were harvested and counted. The total number of fruit per plant
Cultures of Botrytis cinerea and Alternaria alternata (Florida isolate FLc 1) were grown on potato-dextrose-agar plates at room temperature and under room lighting for 7 days. Suspensions of spores were prepared in water and were diluted to a density of approximately 10 000 per ml. Fruit were harvested at the pink (30–70% red) ripening stage, sorted into batches of eight to 12 and ripened off the vine to the light red ripening stage. Each fruit was wounded twice on the shoulder with a 26.5 gauge needle, then each wound site
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was inoculated with 10 ml of spore suspension (containing approximately 100 spores, of which 20– 50 were viable). Fruit were incubated in covered plastic trays, misted on the inside with water, for 4 days at 15 °C, then moved to room temperature. Lesion diameters were measured twice a week and used to calculate lesion area. Experiments consisted of three randomized blocks (each of eight to 12 fruit) per genotype, and were repeated twice per pathogen.
2.5. Fruit processing Fruit was processed using a microwave break as described by Wolcott et al. (1987), with modifications. Batches of approximately 800 g of red ripe fruit were harvested and weighed. Fruit were diced into 1-cm cubes, placed in a large preweighed glass dish and covered with plastic wrap. Diced fruit was microwaved in three steps at full power, for 5 min then contents were stirred, for 3 min then contents were stirred again, then for 2 min. The dish was placed in a large ice bath to cool, then brought to room temperature and returned to the original weight by adding water. Microwaved fruit was forced using a flat plastic spatula through two metal sieves, with meshes of 2 and 0.8 mm, respectively, and retained seeds and skins were discarded. The juice was weighed and soluble solids content determined by refractometry (reported as °Brix), then centrifuged in pre-weighed centrifuge bottles at 4225× g for 10 min to pellet insoluble particulates. The serum was decanted and the bottles were drained upside down, then the bottles containing the pellets were re-weighed and the weight of the insoluble material calculated. Weights of original juice and of the pellet after centrifugation were used to calculate the precipitate weight ratio as a percentage as previously described (Takada and Nelson, 1983). For measurements of serum viscosity, aliquots of serum were re-centrifuged at 10 000× g for 20 min, and the supernatants adjusted to 5.5 °Brix. Serum viscosity of 1 ml aliquots was determined using a 200 mm Cannon – Manning semi-micro viscometer (Fisher Scientific, Pittsburgh, PA) in a water bath at room temperature. For an estimation of paste viscosity, pelleted juice particulates
were resuspended in 15% (by weight) of the serum to form a thick paste, and if necessary adjusted to 5.3 °Brix by dilution. Paste viscosity was measured using a Bostwick consistometer (CSC Scientific, Fairfax, VA), measuring the distance moved (mean of minimum and maximum) by the paste front in 30 s.
2.6. Statistical analysis Statistical comparisons were carried out using the JMP statistical package, version 3.2.2 (SAS Institute, Cary, NC). Experiments were examined first using analysis of variance, then individual means were compared using either Tukey– Kramer or contrast analysis. A significance value of PB 0.05 was used unless otherwise stated.
3. Results
3.1. Agronomic properties Suppression of LeExp1 mRNA and protein accumulation in two independent transgenic lines had no significant effect on the size of ripe fruit, as determined by fruit weight (Fig. 1A). Based on a comparison of the means by Tukey–Kramer analysis, in neither line was transgenic fruit significantly different in size from its corresponding azygous control. The only significant differences in fruit size, between wild-type controls and transgenic fruit of line 17-42-5, and between transgenic fruit of line 17-42-5 and transgenic fruit of line 18-40-11, were thus probably due to variation between particular lines rather than to the presence of a transgene. The number of fruit produced by a mature plant with no further developing flowers was used as an estimate of fruit set (Fig. 1B). Plants of line 18-40, either suppressed in expression of LeExp1 (18-40-11) or azygous, produced significantly more fruit than both plants of line 17-42, either suppressed in expression of LeExp1 (17-42-5) or azygous, or wild-type controls. Based on a comparison of the means by Tukey–Kramer analysis, in both lines 17-42 and 18-40 the transgenic line possessing an LeExp1 transgene was not signifi-
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cantly different from corresponding azygous controls, suggesting that any differences between genotypes were due not to the presence of the transgene but to divergence between lines, possibly the result of somaclonal variation introduced by the tissue culture process.
3.2. Shelf life Previous studies have shown that LeExp1-suppressed fruit of line 18-40 were significantly firmer than corresponding azygous controls throughout ripening, whereas fruit of line 17-42, which were less effectively suppressed in LeExp1 protein accumulation, were significantly firmer than corresponding azygous controls only early in ripening
Fig. 1. Effect of suppression of LeExp1 on fruit weight and total number of fruit per plant. (A) Fruit weight. Data are means9S.E. of measurements from a minimum of 150 fruit per genotype. (B) Number of fruit per mature plant. Data are means9S.E. of 16 plants per genotype. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. Lower case letters show significance groups (PB 0.05).
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Table 1 Scale used to quantify fruit deterioration during storage Score
Deterioration
Description
4
Very severe
3 2
Severe Moderate
1
Slight
0
None
Large regions of extreme softness, wateriness under the skin, wrinkling, fungal infection Regions of extreme softness Small patches of moderate softness Some softness, either uniform or in patches Uniformly firm
For each replicate, individual fruit were assigned to one of the five categories, then the mean score per replicate calculated. All fruit had a deterioration index score of 0 at packing. Fruit developing fungal infections were scored as 4 and removed from the experiment, but scored as 4 at subsequent time points.
(Brummell et al., 1999b). To examine if improved fruit firmness resulted in increased fruit shelf life, two experiments using different packaging materials were carried out. Fruit were packed at the breaker to early pink ripening stage in plastic liners in cardboard shipping boxes to approximate fruit packaging and shipping, and fruit were packed at the red ripe ripening stage in plastic clam shells to mimic retail display conditions. Fruit were assessed at intervals for deterioration on a five-point scale (Table 1). At packing, all fruit had a score of 0. Fruit were examined 35 days after packing in shipping boxes (Fig. 2A). When stored at 13 °C, breaker to pink ripening stage fruit achieved full ripeness approximately 11 days after packing, so examination was approximately 3 weeks after full ripeness. Tukey–Kramer analysis found that LeExp1-suppressed fruit of line 18-40 showed significantly less deterioration, and thus improved shelf life, relative to azygous controls and to wild type fruit. LeExp1-suppressed fruit of line 17-42, which is less suppressed in LeExp1 protein accumulation than line 18-40, was not significantly different from controls. When packed in clam shells stored at 13 °C and examined 10 days after packing, again only LeExp1-suppressed fruit of line 18-40 showed significantly less deterioration than controls (Fig. 2B). Examination at earlier and later
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times found that fruit of line 17-42-5 were always slightly less deteriorated than controls, but the differences were not statistically significant (data not shown). Thus, in the two transgenic lines increased suppression of LeExp1 protein accumulation was correlated with both increased fruit firmness and increased fruit shelf life. Fruit of line 18-40 were used for a time course of fruit quality with time from packing in two different packaging materials (Fig. 3). In liners in shipping boxes, detectable deterioration began ap-
Fig. 3. Effect of suppression of LeExp1 on fruit shelf life with time. (A) Fruit stored in liners in shipping boxes at 13 °C. Fruit were packed at the breaker to early pink ripening stage, and became fully ripe approximately 11 days after packing. Data are means 9 S.E. of six replicates each of eight fruit per genotype. (B) Fruit stored in plastic clam shells at 13 °C. Fruit were packed at the red ripe ripening stage. Data are means 9 S.E. of three replicates each of eight or nine fruit per genotype. Fruit were assessed for deterioration on a five-point scale (see Table 1). Transgenic fruit suppressed in expression of LeExp1 are shown as open symbols and dashed lines, corresponding azygous segregant controls as solid symbols and solid lines.
Fig. 2. Effect of suppression of LeExp1 on fruit shelf life. (A) Fruit stored in liners in shipping boxes at 13 °C for 35 days. Fruit were packed at the breaker to early pink ripening stage, and became fully ripe approximately 11 days after packing. Data are means 9 S.E. of two experiments each consisting of three replicates of eight fruit per genotype. (B) Fruit stored in plastic clam shells at 13 °C for 10 days. Fruit were packed at the red ripe ripening stage. Data are means 9S.E. of three replicates each of eight to 12 fruit per genotype. In both A and B, fruit were assessed for deterioration on a five-point scale (see Table 1). Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. Lower case letters show significance groups (P B 0.05).
proximately 17 days after packing in control (1840-azygous) fruit, whereas this was delayed until 21 days after packing in LeExp1-suppressed 1840-11 fruit (Fig. 3A). The rate of deterioration of control fruit was more rapid than that of LeExp1suppressed fruit, and contrast analysis showed that at all points after 24 days LeExp1-suppressed fruit was significantly less deteriorated than controls (24 days PB0.01, all subsequent times PB 0.001). Fruit packed in clam shells (Fig. 3B) showed much more rapid deterioration than fruit packed in liners in boxes, due firstly to being packed when already ripe, and secondly to bruising of fruit against the internal indentations of the
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packaging and against each other. Nevertheless, LeExp1-suppressed fruit were significantly less deteriorated than controls between 9 and 23 days after packing (PB0.002). Taking a fruit deterioration index of 1.0 as the limit of saleable quality, suppression of LeExp1 protein accumulation increased shelf life of fruit stored at 13 °C from 30 to 40 days in boxes and from 9 to 14 days in clam shells. Such a difference could noticeably reduce packing and retail losses due to wastage.
3.3. Disease resistance Increased firmness of fruit caused by suppression of LeExp1 might be expected to result in improved resistance to postharvest pathogens in addition to improved shelf life. However, the development of disease lesions after infection with B. cinerea (Fig. 4) or A. alternata (Fig. 5) was greater in fruit suppressed in expression of LeExp1 than in corresponding azygous controls or in wild type. This was the case for both of the transgenic lines examined and for both of the
Fig. 5. Development of disease lesions on fruit infected with A. alternata. Fruit were inoculated into a wound with fungal spores, incubated at 15 °C for 4 days then at room temperature, and lesion area measured at intervals. Data for each genotype are means of three replicates each of eight to 12 fruit with two inoculations per fruit. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. A representative experiment (of two) is shown.
postharvest pathogens used. Resistance to Botrytis and Alternaria was thus not improved in LeExp1-suppressed fruit, and contrast analysis showed that resistance was significantly reduced 10 or 13 days postinfection, respectively.
3.4. Processing properties
Fig. 4. Development of disease lesions on fruit infected with B. cinerea. Fruit were inoculated into a wound with fungal spores, incubated at 15 °C for 4 days then at room temperature, and lesion area measured at intervals. Data for each genotype are means of three replicates each of eight to 12 fruit with two inoculations per fruit. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. A representative experiment (of two) is shown.
Juice prepared from fruit of lines 18-40-azygous and 18-40-11 possessed a similar Brix value (a measure of soluble solids content) and a similar precipitate weight ratio (a measure of insoluble solids content) (Table 2). The two genotypes thus also produced a similar weight of serum from a given weight of juice. This suggests that suppression of LeExp1 activity does not substantially affect the amount of insoluble particulates relative to soluble solids in tomato juice. The viscosity of serum was slightly reduced in the LeExp1-suppressed line, as shown by a more rapid efflux time from a viscometer, but the gross viscosity of paste was increased, as shown by reduced movement of the paste in a Bostwick consistometer (Table 2).
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Paste gross viscosity was increased by 18.6% relative to the azygous control. Based on a one-way analysis of variance, the difference between the viscosity of paste from 18-40-11 and from 18-40azygous controls was highly significant (P B 0.0001). Since the paste was prepared by resuspending all the particulates in 15% of the serum, and the viscosity of the serum derived from LeExp1-suppressed fruit was less than that of controls, this increase in gross viscosity was presumably due largely to the influence of the insoluble materials. Thus, suppression of LeExp1 would appear to increase the viscosity of paste by an effect on the characteristics of the particulates.
4. Discussion Suppression of LeExp1 mRNA and protein accumulation did not affect either the final size achieved by ripe fruit, or fruit set as estimated by fruit number per plant (Fig. 1). LeExp1 mRNA and protein do not appear until around the breaker stage when fruit are already full size (Rose et al., 1997, 2000; Brummell et al., 1999a,b), suggesting that LeExp1 protein is not involved in fruit growth. Also, the presence of an LeExp1 transgene in LeExp1-suppressed fruit does not affect the mRNA accumulation of several other members of the expansin gene family which are expressed in expanding green fruit, and which may be involved in fruit growth (Brummell and
Harpster, 2001). Suppression of PME activity also had no significant effects on either fruit size or fruit set (Tieman et al., 1995), and suppression of PG activity did not reduce total fruit yield (Schuch et al., 1991). The suppression of LeExp1 protein accumulation in transgenic tomato fruit resulted in a significant reduction in fruit softening throughout ripening in the best line (line 18-40), and this increase in wall firmness was accompanied by reduced depolymerization of cell wall polyuronides (Brummell et al., 1999b). This difference in pectin metabolism was probably due to an indirect effect of reduced LeExp1 protein activity in the wall, since LeExp1 itself is not thought to act directly on polyuronides, whose disassembly in fruit ripening is largely brought about by the action of PG (Giovannoni et al., 1989). Suppression of PG activity in tomato fruit using transformation by antisense transgenes (Sheehy et al., 1988; Smith et al., 1988) reduced polyuronide depolymerization during ripening, although the reduction was relatively small (Brummell and Labavitch, 1997). Fruit suppressed in PG accumulation were slightly firmer than controls during ripening, but the difference required careful measurement to be detectable (Langley et al., 1994) and showed variability in different genetic backgrounds and under different growing and handling conditions (Kramer et al., 1992). Nevertheless, PG-suppressed fruit showed less deterioration than controls during storage and per-
Table 2 Properties of juice and paste prepared from fruit suppressed in expression of LeExp1 (18-40-11) and azygous segregant controls (18-40-azygous) Genotype
°Brix
Precipitate weight ratio (%)
Serum viscosity (Efflux time (s))
Paste viscosity (cm in 30 s)
18-40-azy
5.5 9 0.1
20.7 9 0.7
50.9 90.6
7.21 90.29
18-40-11
5.5 9 0.1
20.890.5
46.0 92.0
5.87 90.53
Soluble solids content of juice after a microwave break was measured by refractometry at 20 °C as °Brix (mean 9 S.D. from three preparations). Tomato juice was centrifuged to pellet insoluble particulates, and the precipitate weight ratio determined (mean 9 S.D. of three experiments). Serum viscosity was measured at 5.5 °Brix by viscometry (mean 9S.D. of two experiments, three readings per sample). Paste viscosity was estimated at 5.3 °Brix by resuspending insoluble particulates pelleted by centrifugation in 15% by weight of the serum to form a thick paste, and measuring viscosity with a Bostwick consistometer (mean 9 S.D. of three experiments, four readings per sample).
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formed better than controls after handling and shipping (Schuch et al., 1991; Kramer et al., 1992). This appeared to be due to enhanced textural properties resulting from a reduced breakdown of the middle lamella, which decreased the separation between cells during ripening and as the fruit became over ripe (Langley et al., 1994). In contrast, suppression of PME activity did not affect fruit firmness during normal ripening, but severely reduced shelf life since as fruit became over ripe they lost integrity and disintegrated, possibly due to a reduction in strength of the middle lamella (Tieman and Handa, 1994). LeExp1-suppressed fruit of line 18-40 were 13– 23% firmer than controls, depending upon ripening stage (Brummell et al., 1999b), and shelf life was increased by approximately 10 days in boxes and 5 days in clam shells (see Section 3 and Fig. 3). The reduction in polyuronide depolymerization caused by suppression of LeExp1 was greater than that caused by suppression of PG (cf. Brummell and Labavitch, 1997; Brummell et al., 1999b), suggesting that part of the improvement in shelf life of LeExp1-suppressed fruit was due to reduced pectin breakdown. Whether this resulted in diminished cell separation through effects on the middle lamella remains to be determined. However, the increase in firmness of LeExp1-suppressed fruit cannot all be explained by reduced polyuronide depolymerization. LeExp1 is thought to have a direct effect on some aspect of cell wall loosening during ripening which contributes to fruit softening, probably including increasing the accessibility of depolymerases such as EGases to matrix glycans, with an additional perhaps more indirect effect on the accessibility of a pectinase such as PG to polyuronide substrates. A reduction in both the direct effect of LeExp1 on wall loosening and the indirect effect on pectin disassembly may together be responsible for the substantially improved shelf life of LeExp1-suppressed fruit. In fruit suppressed in the expression of PG, the improved tissue integrity afforded by reduced breakdown of the middle lamella brought about enhanced resistance to the soft-rot pathogens Rhizopus stolonifer and Geotrichum candidum (Kramer et al., 1992). These fungi normally infect
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ripening fruit, preferentially infecting at sites of wounding or damage, as do the two pathogens used in the present study, B. cinerea (grey mold) and A. alternata (black rot). However, fruit suppressed in expression of LeExp1, though significantly firmer than controls, were not more resistant to attack by B. cinerea or A. alternata (Figs. 4 and 5). Indeed, these fruit were significantly more susceptible to the pathogens examined, although why this should be is unclear. B. cinerea and A. alternata are both necrotrophic pathogens, having a different effect on the host than the parasitic R. stolonifer and G. candidum, and it was noted that PG-suppressed fruit were not more resistant to B. cinerea (Kramer et al., 1992). The disease symptoms of maceration and cell death caused by necrotrophic pathogens are believed to be brought about by endo-acting pectinases produced by the pathogen (Chesson, 1980; Collmer and Keen, 1986). These enzymes can include various pectin and pectate lyases and polygalacturonases (Mendgen et al., 1996), and the presence of plant polygalacturonide can increase the rate of production of these activities by the microbe (Chatterjee et al., 1979; Johnston and Williamson, 1992). In LeExp1-suppressed fruit, the cell wall polygalacturonides were less depolymerized than in controls, and it is perhaps possible that the higher molecular weight of the pectin substrate or the altered availability of pectin in the wall induced higher rates of pectinase production by the pathogens, resulting in increased disease symptoms. Alternatively, some aspect of ripening-related wall modification that is reduced in LeExp1-suppressed fruit may make the plant cell wall more susceptible to fungal colonization. It thus follows that since fruit suppressed in LeExp1 protein accumulation were more susceptible to pathogen infection, LeExp1 protein and the changes brought about in the wall by its action contribute to resistance to pathogenic infection. The production of tomato juice and paste from fresh fruit requires treatment at high temperature to inactivate endogenous PG activity. PG is very thermostable, and even after heating at 93 °C (a hot break) some PG activity is still detectable in juice (Luh and Daoud, 1971). Polyuronides are depolymerized to a modest extent in the fruit cell
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wall during ripening, but in vitro or during the preparation of juice the limitations on PG action are removed and pectins can be depolymerized to very small size (Seymour et al., 1987; Brummell and Labavitch, 1997). Thus, if PG is not inactivated, the depolymerization of pectin molecules occurring during juice manufacture can result in a substantial loss of juice or paste viscosity (Luh and Daoud, 1971; Bhasin and Bains, 1987). The antisense suppression of PG activity in transgenic fruit resulted in enhanced gross viscosity of tomato juice prepared using a raw cold break, since in controls PG was not inactivated and pectins became extensively degraded (Schuch et al., 1991). When juice was prepared using a microwave hot break, no difference in viscosity due to PG suppression was observed, since the hot break effectively inactivated PG and prevented polyuronide depolymerization even in controls (Schuch et al., 1991). Under usual processing conditions, PG-suppressed lines showed an increase in serum viscosity of up to two-fold (Kramer et al., 1992; Brummell and Labavitch, 1997), suggesting that the observed increases in gross viscosity were due to an increase in the viscosity of the soluble components of the juice. Similarly, in transgenic lines suppressed in PME activity, serum viscosity, whole juice viscosity, paste viscosity and serum separation were all improved relative to controls (Thakur et al., 1996a). Pectins from PME-suppressed lines had a much higher degree of methoxylation than controls, and since pectins that have not been demethylesterified by PME are inaccessible to PG (Tieman et al., 1992), they were protected from depolymerization during juice preparation (Thakur et al., 1996b). This resulted in an increase in serum viscosity of up to 3.5-fold in PME-suppressed lines, depending on processing conditions (Thakur et al., 1996a). In LeExp1-suppressed fruit, paste gross viscosity was substantially increased (by almost 20%) although serum viscosity was not improved relative to controls (Table 2). This suggests that the enhancement in viscosity of paste was due to a change in the properties of the insoluble, particulate components, or on the interaction of the
insoluble material with the soluble serum. However, it should be cautioned that the method used to prepare paste will over-estimate the contribution of the particulates to paste viscosity by increasing interparticle interaction, which affects viscosity (Beresovsky et al., 1995). The increase in wall firmness due to suppression of LeExp1 activity may affect the shape, size or configuration of cell wall fragments, including cellulose microfibrils, in the juice. The form and amount of cell wall fragments, which are approximately half of the insoluble solids, are a very important contributor to rheological properties and are essential for high viscosity in juice or paste (Whittenberger and Nutting, 1957, 1958; Tanglertpaibul and Rao, 1987). It would be very interesting to cross together LeExp1- and PGsuppressed transgenic lines and examine juice and paste prepared from fruit deficient in both activities. The increased viscosity of the particulates caused by suppression of LeExp1 combined with the increased viscosity of the serum caused by suppression of PG might produce a paste with exceptional rheological properties.
Acknowledgements We thank Carol McGugin for assistance with pathogen assays, Minh Ho for fruit weight measurements, Jacquie de Silva and Montan˜ a Ca´ mara for advice on laboratory juice and paste preparation, and Kristen Natoli, Vinnie Pirozzi and Veronica Ochoa for care and maintenance of plants. This work was supported by Seminis Vegetable Seeds.
References Beresovsky, N., Kopelman, I.J., Mizrahi, S., 1995. The role of pulp interparticle interaction in determining tomato juice viscosity. J. Food Process. Preserv. 19, 133 – 146. Bhasin, U.R., Bains, G.S., 1987. Study of pectolytic factors and processing in relation to rheological characteristics of tomato juice. J. Food Sci. Technol. 24, 247 – 253. Brummell, D.A., Harpster, M.H., 2001. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. Biol. 47, 311 – 340.
D.A. Brummell et al. / Posthar6est Biology and Technology 25 (2002) 209–220 Brummell, D.A., Labavitch, J.M., 1997. Effect of antisense suppression of endopolygalacturonase activity on polyuronide molecular weight in ripening tomato fruit and in fruit homogenates. Plant Physiol. 115, 717 –725. Brummell, D.A., Harpster, M.H., Dunsmuir, P., 1999a. Differential expression of expansin gene family members during growth and ripening of tomato fruit. Plant Mol. Biol. 39, 161 – 169. Brummell, D.A., Harpster, M.H., Civello, P.M., Palys, J.M., Bennett, A.B., Dunsmuir, P., 1999b. Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. Plant Cell 11, 2203 – 2216. Catala, C., Rose, J.K.C., Bennett, A.B., 2000. Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth. Plant Physiol. 122, 527 – 534. Chatterjee, A.K., Buchanan, G.E., Behrens, M.K., Starr, M.P., 1979. Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species. Can. J. Microbiol. 25, 94 –102. Chesson, A., 1980. Maceration in relation to the post-harvest handling and processing of plant material. J. Appl. Bacteriol. 48, 1 – 45. Cho, H.-T., Cosgrove, D.J., 2000. Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 97, 9783 – 9788. Collmer, A., Keen, N., 1986. The role of pectic enzymes in plant pathogenesis. Annu. Rev. Phytopathol. 24, 383 –409. Cosgrove, D.J., 2000. Loosening of plant cell walls by expansins. Nature 407, 321 –326. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., Fischer, R.L., 1989. Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fruit results in polyuronide degradation but not fruit softening. Plant Cell 1, 53 – 63. Johnston, D.J., Williamson, B., 1992. Purification and characterization of four polygalacturonases from Botrytis cinerea. Mycol. Res. 96, 343 –349. Kramer, M., Sanders, R., Bolkan, H., Waters, C., Sheehy, R.E., Hiatt, W.R., 1992. Postharvest evaluation of transgenic tomatoes with reduced levels of polygalacturonase: processing, firmness and disease resistance. Postharvest Biol. Technol. 1, 241 –255. Langley, K.R., Martin, A., Stenning, R., Murray, A.J., Hobson, G.E., Schuch, W.W., Bird, C.R., 1994. Mechanical and optical assessment of the ripening of tomato fruit with reduced polygalacturonase activity. J. Sci. Food Agric. 66, 547 – 554. Luh, B.S., Daoud, H.N., 1971. Effect of break temperature and holding time on pectin and pectic enzymes in tomato pulp. J. Food Sci. 36, 1039 –1043. Maclachlan, G., Brady, C., 1994. Endo-1,4-b-glucanase, xyloglucanase and xyloglucan endo-transglycosylase activities versus potential substrates in ripening tomatoes. Plant Physiol. 105, 965 – 974.
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McQueen-Mason, S., Durachko, D.M., Cosgrove, D.J., 1992. Two endogenous proteins that induce cell wall extension in plants. Plant Cell 4, 1425 – 1433. Mendgen, K., Hahn, M., Deising, H., 1996. Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu. Rev. Phytopathol. 34, 367 – 386. Rose, J.K.C., Lee, H.H., Bennett, A.B., 1997. Expression of a divergent expansin gene is fruit-specific and ripening-regulated. Proc. Natl. Acad. Sci. USA 94, 5955 – 5960. Rose, J.K.C., Cosgrove, D.J., Albersheim, P., Darvill, A.G., Bennett, A.B., 2000. Detection of expansin proteins and activity during tomato fruit ontogeny. Plant Physiol. 123, 1583 – 1592. Schuch, W., Kanczler, J., Robertson, D., Hobson, G., Tucker, G., Grierson, D., Bright, S., Bird, C., 1991. Fruit quality characteristics of transgenic tomato fruit with altered polygalacturonase activity. HortScience 26, 1517 – 1520. Seymour, G.B., Lasslett, Y., Tucker, G.A., 1987. Differential effects of pectolytic enzymes on tomato polyuronides in vivo and in vitro. Phytochemistry 26, 3137 – 3139. Shcherban, T.Y., Shi, J., Durachko, D.M., Guiltinan, M.J., McQueen-Mason, S.J., Shieh, M., Cosgrove, D.J., 1995. Molecular cloning and sequence analysis of expansins-a highly conserved, multigene family of proteins that mediate cell wall extension in plants. Proc. Natl. Acad. Sci. USA 92, 9245 – 9249. Sheehy, R.E., Kramer, M., Hiatt, W.R., 1988. Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc. Natl. Acad. Sci. USA 85, 8805 – 8809. Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.R., Morris, P.C., Schuch, W., Grierson, D., 1988. Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334, 724 – 726. Takada, N., Nelson, P.E., 1983. A new consistency method for tomato products: the precipitate weight ratio. J. Food Sci. 48, 1460 – 1462. Tanglertpaibul, T., Rao, M.A., 1987. Rheological properties of tomato concentrates as affected by particle size and methods of concentration. J. Food Sci. 52, 141 – 145. Thakur, B.R., Singh, R.K., Tieman, D.M., Handa, A.K., 1996a. Tomato product quality from transgenic fruits with reduced pectin methylesterase. J. Food Sci. 61, 85 – 108. Thakur, B.R., Singh, R.K., Handa, A.K., 1996b. Effect of an antisense pectin methylesterase gene on the chemistry of pectin in tomato (Lycopersicon esculentum) juice. J. Agric. Food Chem. 44, 628 – 630. Thakur, B.R., Singh, R.K., Nelson, P.E., 1996c. Quality attributes of processed tomato products: a review. Food Rev. Int. 12, 375 – 401. Tieman, D.M., Handa, A.K., 1994. Reduction in pectin methylesterase activity modifies tissue integrity and cation levels in ripening tomato (Lycopersicon esculentum Mill.) fruits. Plant Physiol. 106, 429 – 436. Tieman, D.M., Harriman, R.W., Ramamohan, G., Handa, A.K., 1992. An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit. Plant Cell 4, 667 – 679.
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Tieman, D.M., Kausch, K.D., Serra, D.M., Handa, A.K., 1995. Field performance of transgenic tomato with reduced pectin methylesterase activity. J. Am. Soc. Hortic. Sci. 120, 765– 770. Tong, C.B.S., Gross, K.C., 1988. Glycosyl-linkage composition of tomato fruit cell wall hemicellulosic fractions during ripening. Physiol. Plant 74, 365 –370. Whittenberger, R.T., Nutting, G.C., 1957. Effect of tomato
cell structures on consistency of tomato juice. Food Technol. 11, 19 – 22. Whittenberger, R.T., Nutting, G.C., 1958. High viscosity of cell wall suspensions prepared from tomato juice. Food Technol. 12, 420 – 424. Wolcott, T., Marsh, G.L., Merson, R.L., 1987. Methods for rapidly evaluating consistency potential of new processing tomato varieties. Acta Hortic. 200, 115 – 124.
Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin David A. Brummell *, William J. Howie, Christa Ma, Pamela Dunsmuir DNA Plant Technology, 6701 San Pablo A6enue, Oakland, CA 94608, USA Received 25 July 2001; accepted 6 October 2001
Abstract Expansins are proteins that cause cell wall loosening, and are involved in many aspects of cell wall modification during development. In tomato, the expansin gene LeExp1 shows ripening-related accumulation of mRNA and protein, and transgenic silencing of the expression of this gene results in tomato fruit that are significantly firmer than corresponding azygous controls throughout ripening. Examination of postharvest quality characteristics of fruit suppressed in accumulation of LeExp1 protein found that increased firmness resulted in significantly improved fruit integrity during storage at 13 °C. Based upon the first appearance of noticeable deterioration, fruit shelf life was extended by 5–10 days, depending upon the packaging. However, the increased firmness of LeExp1-suppressed fruit did not result in increased resistance to the necrophytic pathogens Botrytis cinerea and Alternaria alternata. Juice prepared from LeExp1-suppressed fruit following a microwave break had a soluble solids content (°Brix), insoluble solids content (precipitate weight ratio) and serum viscosity similar to controls. Resuspension of the insoluble pelleted particulate material in 15% of the serum produced a thick paste, which allowed estimation of gross viscosity in a Bostwick consistometer. The viscosity of paste from LeExp1-suppressed fruit was 19% greater than that from corresponding azygous controls, presumably due to changes in the insoluble particulate components affecting flow characteristics. No significant effects of the LeExp1 transgene on fruit size or fruit number per plant were noted. The data suggest that fruit suppressed in expression of LeExp1 have improved shelf life and processing properties. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Expansin; Fruit quality; Processing; Ripening; Shelf life; Tomato
1. Introduction Fruit quality has many aspects, including not only the obvious properties of flavor, color, nutri* Corresponding author. Present address: Department of Pomology, University of California, Davis, CA 95616, USA. Tel.: +1-530-752-3971; fax: + 1-530-752-8502. E-mail address: [email protected] (D.A. Brummell).
tional content and firmness, but also shelf life, processing qualities and resistance to pre- and postharvest pathogens. Excessive softening is the main factor responsible for the deterioration that limits shipping, storage and saleability, and fruit firmness and texture also affect the integrity of chopped and diced fruit used for canning and fruit products. In the case of tomato, firmness and texture influence the viscosity of processed juice
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and paste, which is a major component of value (Thakur et al., 1996c). Reducing fruit softening during ripening enhances shelf life and reduces spoilage and waste, and is of great commercial interest. Fruit softening during ripening is brought about by a disassembly of the fruit cell wall, the major changes including depolymerization and solubilization of pectins, depolymerization of matrix glycans (hemicelluloses), and loss of pectic galactose side chains (Brummell and Harpster, 2001). The cell wall-modifying proteins that bring about changes in cell wall structure and its component polysaccharides have not all been identified, but transgenic suppression of ripening-related enzymes involved in pectin metabolism resulted in improvements in various aspects of quality. Suppression of polygalacturonase (PG) activity caused only a very small reduction in fruit softening with ripening, but extended fruit shelf life, reduced fruit susceptibility to postharvest pathogens and increased the viscosity of juice and paste prepared from these fruit (Schuch et al., 1991; Kramer et al., 1992; Langley et al., 1994). Similarly, transgenic suppression of pectin methylesterase (PME) activity had little effect on fruit softening during ripening, but processed juice and paste showed higher soluble solids content and increased viscosity (Tieman and Handa, 1994; Tieman et al., 1992, 1995; Thakur et al., 1996a). Thus, increased fruit quality can result from reduced cell wall breakdown during ripening, not necessarily an improvement in firmness but an improvement in fruit integrity and in the properties of processed fruit products. The matrix glycan components of the cell wall also become depolymerized during ripening (Tong and Gross, 1988; Maclachlan and Brady, 1994), and this is a major contributor to fruit softening. Enzymes which might be involved in the breakdown of matrix glycans include endo-1,4-b-glucanases (EGases), xyloglucan endotransglycosylases and expansins (Brummell and Harpster, 2001). Expansins are cell wall-localized proteins that possess a structure with similarities to carbohydrate-binding proteins (Shcherban et al., 1995), and are believed to act in the wall by disrupting the non-covalent binding between matrix glycans
and cellulose microfibrils (Cosgrove, 2000). They are encoded by large gene families, consisting of at least 24 genes in Arabidopsis (Cosgrove, 2000). Expansins bring about the cell wall loosening responsible for growth (McQueen-Mason et al., 1992), and several expansin gene family members are expressed in expanding and maturing green tomato fruit (Brummell et al., 1999a; Catala et al., 2000). Expansins are also involved in other developmental processes such as abscission (Cho and Cosgrove, 2000), and in tomato the mRNA of one expansin gene, LeExp1, was found to accumulate specifically during fruit ripening and showed the regulation by ethylene characteristic of ripening-related genes in climacteric species (Rose et al., 1997). Suppression of LeExp1 mRNA and protein accumulation in transgenic fruit reduced softening during ripening, whereas overexpression of LeExp1 mRNA and protein increased fruit softening (Brummell et al., 1999b). Modification of LeExp1 protein levels in fruit was found to result in changes in cell wall polymer metabolism, and it appears LeExp1 is an important component of the cell wall-modifying machinery. Since suppression of the ripening-related expansin LeExp1 reduced fruit softening during ripening, the aim of this work was to examine if improved fruit firmness also resulted in enhancements to other aspects of fruit quality, such as fruit shelf life, disease resistance and the viscosity of processed products. Two transgenic lines were selected from two large populations of primary transformants possessing LeExp1 transgenes under the control of the constitutive cauliflower mosaic virus 35S promoter (Brummell et al., 1999b). Primary transformant 17-42 was transformed with a complete, functional LeExp1 open reading frame, whereas line 18-40 was transformed with a truncated version that could not produce functional protein from the transgene. These two lines showed strong silencing of both the endogenous LeExp1 gene and the transgene, and in red ripe fruit homozygous progeny possessed approximately 5 and 3%, respectively, of wild type levels of immunodetectable LeExp1 protein (Brummell et al., 1999b). Seeds from these lines homozygous for the transgene were the transgenic material used in this study.
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2. Materials and methods
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(including previously-harvested and dropped fruit) was used as an estimate of fruit set.
2.1. Plant material 2.3. Shelf life Tomato (Lycopersicon esculentum Mill., cv ‘Ailsa Craig’) plants suppressed in the expression of the ripening-related expansin gene LeExp1 were generated by constitutive expression of sense LeExp1 transgenes as previously described (Brummell et al., 1999b). Primary transformants (T1 generation) 17-42 and 18-40 showing strong suppression of LeExp1 mRNA and protein accumulation were allowed to self-fertilize, and seeds were collected from homozygous T2 generation plants 17-42-5 and 18-40-11, and from corresponding azygous sibling plants. Two populations of T3 generation plants (each consisting of 16 plants of each genotype and 16 wild type controls) were planted in September 1998 and February 1999, respectively, and were grown to maturity in a greenhouse. The first population was used for studies of yield characteristics and disease resistance, and the second for evaluation of shelf life. A third population of 40 plants each of genotypes 18-40-11 and 18-40-azygous for a larger-scale repeat of shelf life properties and for fruit juice and paste production was planted in May 1999. Thus for each line (either 17-42 or 18-40) there was a homozygous transgenic population (progeny of 17-42-5 or 18-40-11) and an azygous control population that was no longer transgenic, since the transgene had been eliminated by segregation, but the plants of which were derived from the same cell line during tissue culture.
Fruit were harvested at breaker (the first appearance of red color on the exterior of the fruit), turning (up to 10% red) or early pink (10–30% red) ripening stages, and surface washed in large volumes of water containing 2.5 ml commercial bleach per liter, then rinsed with tap water. Fruit were blotted then air dried and sorted, and fruit with any abnormalities or damage were discarded. For packing in boxes, fruit were packed at this ripening stage. Preliminary experiments consisted of three replicates each of eight fruit per genotype, packed in random blocks in flat cardboard shipping boxes containing plastic liners holding 25 (5× 5) fruit. Boxes were stored at 13 °C. For packing in plastic clam shells, fruit were ripened at room temperature until red ripe (approximately 7 days after harvest) before packing. A preliminary experiment consisted of three clam shells filled with fruit (eight to 12 fruit, depending on fruit size) per genotype. Clam shells were stored at 13 °C. Fruit were assessed for deterioration on a five-point scale (see Section 3) at intervals. Subsequent experiments used the 18-40-11 and 18-40-azygous genotypes only. For boxes, six replicates each consisting of eight fruit were used per genotype. For clam shells, three replicates each consisting of eight or nine fruit were used per genotype. Fruit were assessed for deterioration on a five-point scale twice per week.
2.2. Agronomic properties
2.4. Disease resistance
For measurements of fruit size, initial experiments found that fruit diameter and fruit weight were directly correlated (data not shown), suggesting that none of the genotypes had an effect on fruit shape. Thereafter, only fruit weight was recorded, and data shown are means based on measurements from a minimum of 150 fruit per genotype. All fruit harvested from each plant were recorded. When plants were mature and all flowers had set fruit, remaining fruit were harvested and counted. The total number of fruit per plant
Cultures of Botrytis cinerea and Alternaria alternata (Florida isolate FLc 1) were grown on potato-dextrose-agar plates at room temperature and under room lighting for 7 days. Suspensions of spores were prepared in water and were diluted to a density of approximately 10 000 per ml. Fruit were harvested at the pink (30–70% red) ripening stage, sorted into batches of eight to 12 and ripened off the vine to the light red ripening stage. Each fruit was wounded twice on the shoulder with a 26.5 gauge needle, then each wound site
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was inoculated with 10 ml of spore suspension (containing approximately 100 spores, of which 20– 50 were viable). Fruit were incubated in covered plastic trays, misted on the inside with water, for 4 days at 15 °C, then moved to room temperature. Lesion diameters were measured twice a week and used to calculate lesion area. Experiments consisted of three randomized blocks (each of eight to 12 fruit) per genotype, and were repeated twice per pathogen.
2.5. Fruit processing Fruit was processed using a microwave break as described by Wolcott et al. (1987), with modifications. Batches of approximately 800 g of red ripe fruit were harvested and weighed. Fruit were diced into 1-cm cubes, placed in a large preweighed glass dish and covered with plastic wrap. Diced fruit was microwaved in three steps at full power, for 5 min then contents were stirred, for 3 min then contents were stirred again, then for 2 min. The dish was placed in a large ice bath to cool, then brought to room temperature and returned to the original weight by adding water. Microwaved fruit was forced using a flat plastic spatula through two metal sieves, with meshes of 2 and 0.8 mm, respectively, and retained seeds and skins were discarded. The juice was weighed and soluble solids content determined by refractometry (reported as °Brix), then centrifuged in pre-weighed centrifuge bottles at 4225× g for 10 min to pellet insoluble particulates. The serum was decanted and the bottles were drained upside down, then the bottles containing the pellets were re-weighed and the weight of the insoluble material calculated. Weights of original juice and of the pellet after centrifugation were used to calculate the precipitate weight ratio as a percentage as previously described (Takada and Nelson, 1983). For measurements of serum viscosity, aliquots of serum were re-centrifuged at 10 000× g for 20 min, and the supernatants adjusted to 5.5 °Brix. Serum viscosity of 1 ml aliquots was determined using a 200 mm Cannon – Manning semi-micro viscometer (Fisher Scientific, Pittsburgh, PA) in a water bath at room temperature. For an estimation of paste viscosity, pelleted juice particulates
were resuspended in 15% (by weight) of the serum to form a thick paste, and if necessary adjusted to 5.3 °Brix by dilution. Paste viscosity was measured using a Bostwick consistometer (CSC Scientific, Fairfax, VA), measuring the distance moved (mean of minimum and maximum) by the paste front in 30 s.
2.6. Statistical analysis Statistical comparisons were carried out using the JMP statistical package, version 3.2.2 (SAS Institute, Cary, NC). Experiments were examined first using analysis of variance, then individual means were compared using either Tukey– Kramer or contrast analysis. A significance value of PB 0.05 was used unless otherwise stated.
3. Results
3.1. Agronomic properties Suppression of LeExp1 mRNA and protein accumulation in two independent transgenic lines had no significant effect on the size of ripe fruit, as determined by fruit weight (Fig. 1A). Based on a comparison of the means by Tukey–Kramer analysis, in neither line was transgenic fruit significantly different in size from its corresponding azygous control. The only significant differences in fruit size, between wild-type controls and transgenic fruit of line 17-42-5, and between transgenic fruit of line 17-42-5 and transgenic fruit of line 18-40-11, were thus probably due to variation between particular lines rather than to the presence of a transgene. The number of fruit produced by a mature plant with no further developing flowers was used as an estimate of fruit set (Fig. 1B). Plants of line 18-40, either suppressed in expression of LeExp1 (18-40-11) or azygous, produced significantly more fruit than both plants of line 17-42, either suppressed in expression of LeExp1 (17-42-5) or azygous, or wild-type controls. Based on a comparison of the means by Tukey–Kramer analysis, in both lines 17-42 and 18-40 the transgenic line possessing an LeExp1 transgene was not signifi-
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cantly different from corresponding azygous controls, suggesting that any differences between genotypes were due not to the presence of the transgene but to divergence between lines, possibly the result of somaclonal variation introduced by the tissue culture process.
3.2. Shelf life Previous studies have shown that LeExp1-suppressed fruit of line 18-40 were significantly firmer than corresponding azygous controls throughout ripening, whereas fruit of line 17-42, which were less effectively suppressed in LeExp1 protein accumulation, were significantly firmer than corresponding azygous controls only early in ripening
Fig. 1. Effect of suppression of LeExp1 on fruit weight and total number of fruit per plant. (A) Fruit weight. Data are means9S.E. of measurements from a minimum of 150 fruit per genotype. (B) Number of fruit per mature plant. Data are means9S.E. of 16 plants per genotype. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. Lower case letters show significance groups (PB 0.05).
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Table 1 Scale used to quantify fruit deterioration during storage Score
Deterioration
Description
4
Very severe
3 2
Severe Moderate
1
Slight
0
None
Large regions of extreme softness, wateriness under the skin, wrinkling, fungal infection Regions of extreme softness Small patches of moderate softness Some softness, either uniform or in patches Uniformly firm
For each replicate, individual fruit were assigned to one of the five categories, then the mean score per replicate calculated. All fruit had a deterioration index score of 0 at packing. Fruit developing fungal infections were scored as 4 and removed from the experiment, but scored as 4 at subsequent time points.
(Brummell et al., 1999b). To examine if improved fruit firmness resulted in increased fruit shelf life, two experiments using different packaging materials were carried out. Fruit were packed at the breaker to early pink ripening stage in plastic liners in cardboard shipping boxes to approximate fruit packaging and shipping, and fruit were packed at the red ripe ripening stage in plastic clam shells to mimic retail display conditions. Fruit were assessed at intervals for deterioration on a five-point scale (Table 1). At packing, all fruit had a score of 0. Fruit were examined 35 days after packing in shipping boxes (Fig. 2A). When stored at 13 °C, breaker to pink ripening stage fruit achieved full ripeness approximately 11 days after packing, so examination was approximately 3 weeks after full ripeness. Tukey–Kramer analysis found that LeExp1-suppressed fruit of line 18-40 showed significantly less deterioration, and thus improved shelf life, relative to azygous controls and to wild type fruit. LeExp1-suppressed fruit of line 17-42, which is less suppressed in LeExp1 protein accumulation than line 18-40, was not significantly different from controls. When packed in clam shells stored at 13 °C and examined 10 days after packing, again only LeExp1-suppressed fruit of line 18-40 showed significantly less deterioration than controls (Fig. 2B). Examination at earlier and later
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times found that fruit of line 17-42-5 were always slightly less deteriorated than controls, but the differences were not statistically significant (data not shown). Thus, in the two transgenic lines increased suppression of LeExp1 protein accumulation was correlated with both increased fruit firmness and increased fruit shelf life. Fruit of line 18-40 were used for a time course of fruit quality with time from packing in two different packaging materials (Fig. 3). In liners in shipping boxes, detectable deterioration began ap-
Fig. 3. Effect of suppression of LeExp1 on fruit shelf life with time. (A) Fruit stored in liners in shipping boxes at 13 °C. Fruit were packed at the breaker to early pink ripening stage, and became fully ripe approximately 11 days after packing. Data are means 9 S.E. of six replicates each of eight fruit per genotype. (B) Fruit stored in plastic clam shells at 13 °C. Fruit were packed at the red ripe ripening stage. Data are means 9 S.E. of three replicates each of eight or nine fruit per genotype. Fruit were assessed for deterioration on a five-point scale (see Table 1). Transgenic fruit suppressed in expression of LeExp1 are shown as open symbols and dashed lines, corresponding azygous segregant controls as solid symbols and solid lines.
Fig. 2. Effect of suppression of LeExp1 on fruit shelf life. (A) Fruit stored in liners in shipping boxes at 13 °C for 35 days. Fruit were packed at the breaker to early pink ripening stage, and became fully ripe approximately 11 days after packing. Data are means 9 S.E. of two experiments each consisting of three replicates of eight fruit per genotype. (B) Fruit stored in plastic clam shells at 13 °C for 10 days. Fruit were packed at the red ripe ripening stage. Data are means 9S.E. of three replicates each of eight to 12 fruit per genotype. In both A and B, fruit were assessed for deterioration on a five-point scale (see Table 1). Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. Lower case letters show significance groups (P B 0.05).
proximately 17 days after packing in control (1840-azygous) fruit, whereas this was delayed until 21 days after packing in LeExp1-suppressed 1840-11 fruit (Fig. 3A). The rate of deterioration of control fruit was more rapid than that of LeExp1suppressed fruit, and contrast analysis showed that at all points after 24 days LeExp1-suppressed fruit was significantly less deteriorated than controls (24 days PB0.01, all subsequent times PB 0.001). Fruit packed in clam shells (Fig. 3B) showed much more rapid deterioration than fruit packed in liners in boxes, due firstly to being packed when already ripe, and secondly to bruising of fruit against the internal indentations of the
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packaging and against each other. Nevertheless, LeExp1-suppressed fruit were significantly less deteriorated than controls between 9 and 23 days after packing (PB0.002). Taking a fruit deterioration index of 1.0 as the limit of saleable quality, suppression of LeExp1 protein accumulation increased shelf life of fruit stored at 13 °C from 30 to 40 days in boxes and from 9 to 14 days in clam shells. Such a difference could noticeably reduce packing and retail losses due to wastage.
3.3. Disease resistance Increased firmness of fruit caused by suppression of LeExp1 might be expected to result in improved resistance to postharvest pathogens in addition to improved shelf life. However, the development of disease lesions after infection with B. cinerea (Fig. 4) or A. alternata (Fig. 5) was greater in fruit suppressed in expression of LeExp1 than in corresponding azygous controls or in wild type. This was the case for both of the transgenic lines examined and for both of the
Fig. 5. Development of disease lesions on fruit infected with A. alternata. Fruit were inoculated into a wound with fungal spores, incubated at 15 °C for 4 days then at room temperature, and lesion area measured at intervals. Data for each genotype are means of three replicates each of eight to 12 fruit with two inoculations per fruit. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. A representative experiment (of two) is shown.
postharvest pathogens used. Resistance to Botrytis and Alternaria was thus not improved in LeExp1-suppressed fruit, and contrast analysis showed that resistance was significantly reduced 10 or 13 days postinfection, respectively.
3.4. Processing properties
Fig. 4. Development of disease lesions on fruit infected with B. cinerea. Fruit were inoculated into a wound with fungal spores, incubated at 15 °C for 4 days then at room temperature, and lesion area measured at intervals. Data for each genotype are means of three replicates each of eight to 12 fruit with two inoculations per fruit. Transgenic fruit suppressed in expression of LeExp1 are shown as hatched bars, corresponding azygous segregant controls as solid black bars, wild type ‘Ailsa Craig’ fruit as solid grey bars. A representative experiment (of two) is shown.
Juice prepared from fruit of lines 18-40-azygous and 18-40-11 possessed a similar Brix value (a measure of soluble solids content) and a similar precipitate weight ratio (a measure of insoluble solids content) (Table 2). The two genotypes thus also produced a similar weight of serum from a given weight of juice. This suggests that suppression of LeExp1 activity does not substantially affect the amount of insoluble particulates relative to soluble solids in tomato juice. The viscosity of serum was slightly reduced in the LeExp1-suppressed line, as shown by a more rapid efflux time from a viscometer, but the gross viscosity of paste was increased, as shown by reduced movement of the paste in a Bostwick consistometer (Table 2).
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Paste gross viscosity was increased by 18.6% relative to the azygous control. Based on a one-way analysis of variance, the difference between the viscosity of paste from 18-40-11 and from 18-40azygous controls was highly significant (P B 0.0001). Since the paste was prepared by resuspending all the particulates in 15% of the serum, and the viscosity of the serum derived from LeExp1-suppressed fruit was less than that of controls, this increase in gross viscosity was presumably due largely to the influence of the insoluble materials. Thus, suppression of LeExp1 would appear to increase the viscosity of paste by an effect on the characteristics of the particulates.
4. Discussion Suppression of LeExp1 mRNA and protein accumulation did not affect either the final size achieved by ripe fruit, or fruit set as estimated by fruit number per plant (Fig. 1). LeExp1 mRNA and protein do not appear until around the breaker stage when fruit are already full size (Rose et al., 1997, 2000; Brummell et al., 1999a,b), suggesting that LeExp1 protein is not involved in fruit growth. Also, the presence of an LeExp1 transgene in LeExp1-suppressed fruit does not affect the mRNA accumulation of several other members of the expansin gene family which are expressed in expanding green fruit, and which may be involved in fruit growth (Brummell and
Harpster, 2001). Suppression of PME activity also had no significant effects on either fruit size or fruit set (Tieman et al., 1995), and suppression of PG activity did not reduce total fruit yield (Schuch et al., 1991). The suppression of LeExp1 protein accumulation in transgenic tomato fruit resulted in a significant reduction in fruit softening throughout ripening in the best line (line 18-40), and this increase in wall firmness was accompanied by reduced depolymerization of cell wall polyuronides (Brummell et al., 1999b). This difference in pectin metabolism was probably due to an indirect effect of reduced LeExp1 protein activity in the wall, since LeExp1 itself is not thought to act directly on polyuronides, whose disassembly in fruit ripening is largely brought about by the action of PG (Giovannoni et al., 1989). Suppression of PG activity in tomato fruit using transformation by antisense transgenes (Sheehy et al., 1988; Smith et al., 1988) reduced polyuronide depolymerization during ripening, although the reduction was relatively small (Brummell and Labavitch, 1997). Fruit suppressed in PG accumulation were slightly firmer than controls during ripening, but the difference required careful measurement to be detectable (Langley et al., 1994) and showed variability in different genetic backgrounds and under different growing and handling conditions (Kramer et al., 1992). Nevertheless, PG-suppressed fruit showed less deterioration than controls during storage and per-
Table 2 Properties of juice and paste prepared from fruit suppressed in expression of LeExp1 (18-40-11) and azygous segregant controls (18-40-azygous) Genotype
°Brix
Precipitate weight ratio (%)
Serum viscosity (Efflux time (s))
Paste viscosity (cm in 30 s)
18-40-azy
5.5 9 0.1
20.7 9 0.7
50.9 90.6
7.21 90.29
18-40-11
5.5 9 0.1
20.890.5
46.0 92.0
5.87 90.53
Soluble solids content of juice after a microwave break was measured by refractometry at 20 °C as °Brix (mean 9 S.D. from three preparations). Tomato juice was centrifuged to pellet insoluble particulates, and the precipitate weight ratio determined (mean 9 S.D. of three experiments). Serum viscosity was measured at 5.5 °Brix by viscometry (mean 9S.D. of two experiments, three readings per sample). Paste viscosity was estimated at 5.3 °Brix by resuspending insoluble particulates pelleted by centrifugation in 15% by weight of the serum to form a thick paste, and measuring viscosity with a Bostwick consistometer (mean 9 S.D. of three experiments, four readings per sample).
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formed better than controls after handling and shipping (Schuch et al., 1991; Kramer et al., 1992). This appeared to be due to enhanced textural properties resulting from a reduced breakdown of the middle lamella, which decreased the separation between cells during ripening and as the fruit became over ripe (Langley et al., 1994). In contrast, suppression of PME activity did not affect fruit firmness during normal ripening, but severely reduced shelf life since as fruit became over ripe they lost integrity and disintegrated, possibly due to a reduction in strength of the middle lamella (Tieman and Handa, 1994). LeExp1-suppressed fruit of line 18-40 were 13– 23% firmer than controls, depending upon ripening stage (Brummell et al., 1999b), and shelf life was increased by approximately 10 days in boxes and 5 days in clam shells (see Section 3 and Fig. 3). The reduction in polyuronide depolymerization caused by suppression of LeExp1 was greater than that caused by suppression of PG (cf. Brummell and Labavitch, 1997; Brummell et al., 1999b), suggesting that part of the improvement in shelf life of LeExp1-suppressed fruit was due to reduced pectin breakdown. Whether this resulted in diminished cell separation through effects on the middle lamella remains to be determined. However, the increase in firmness of LeExp1-suppressed fruit cannot all be explained by reduced polyuronide depolymerization. LeExp1 is thought to have a direct effect on some aspect of cell wall loosening during ripening which contributes to fruit softening, probably including increasing the accessibility of depolymerases such as EGases to matrix glycans, with an additional perhaps more indirect effect on the accessibility of a pectinase such as PG to polyuronide substrates. A reduction in both the direct effect of LeExp1 on wall loosening and the indirect effect on pectin disassembly may together be responsible for the substantially improved shelf life of LeExp1-suppressed fruit. In fruit suppressed in the expression of PG, the improved tissue integrity afforded by reduced breakdown of the middle lamella brought about enhanced resistance to the soft-rot pathogens Rhizopus stolonifer and Geotrichum candidum (Kramer et al., 1992). These fungi normally infect
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ripening fruit, preferentially infecting at sites of wounding or damage, as do the two pathogens used in the present study, B. cinerea (grey mold) and A. alternata (black rot). However, fruit suppressed in expression of LeExp1, though significantly firmer than controls, were not more resistant to attack by B. cinerea or A. alternata (Figs. 4 and 5). Indeed, these fruit were significantly more susceptible to the pathogens examined, although why this should be is unclear. B. cinerea and A. alternata are both necrotrophic pathogens, having a different effect on the host than the parasitic R. stolonifer and G. candidum, and it was noted that PG-suppressed fruit were not more resistant to B. cinerea (Kramer et al., 1992). The disease symptoms of maceration and cell death caused by necrotrophic pathogens are believed to be brought about by endo-acting pectinases produced by the pathogen (Chesson, 1980; Collmer and Keen, 1986). These enzymes can include various pectin and pectate lyases and polygalacturonases (Mendgen et al., 1996), and the presence of plant polygalacturonide can increase the rate of production of these activities by the microbe (Chatterjee et al., 1979; Johnston and Williamson, 1992). In LeExp1-suppressed fruit, the cell wall polygalacturonides were less depolymerized than in controls, and it is perhaps possible that the higher molecular weight of the pectin substrate or the altered availability of pectin in the wall induced higher rates of pectinase production by the pathogens, resulting in increased disease symptoms. Alternatively, some aspect of ripening-related wall modification that is reduced in LeExp1-suppressed fruit may make the plant cell wall more susceptible to fungal colonization. It thus follows that since fruit suppressed in LeExp1 protein accumulation were more susceptible to pathogen infection, LeExp1 protein and the changes brought about in the wall by its action contribute to resistance to pathogenic infection. The production of tomato juice and paste from fresh fruit requires treatment at high temperature to inactivate endogenous PG activity. PG is very thermostable, and even after heating at 93 °C (a hot break) some PG activity is still detectable in juice (Luh and Daoud, 1971). Polyuronides are depolymerized to a modest extent in the fruit cell
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wall during ripening, but in vitro or during the preparation of juice the limitations on PG action are removed and pectins can be depolymerized to very small size (Seymour et al., 1987; Brummell and Labavitch, 1997). Thus, if PG is not inactivated, the depolymerization of pectin molecules occurring during juice manufacture can result in a substantial loss of juice or paste viscosity (Luh and Daoud, 1971; Bhasin and Bains, 1987). The antisense suppression of PG activity in transgenic fruit resulted in enhanced gross viscosity of tomato juice prepared using a raw cold break, since in controls PG was not inactivated and pectins became extensively degraded (Schuch et al., 1991). When juice was prepared using a microwave hot break, no difference in viscosity due to PG suppression was observed, since the hot break effectively inactivated PG and prevented polyuronide depolymerization even in controls (Schuch et al., 1991). Under usual processing conditions, PG-suppressed lines showed an increase in serum viscosity of up to two-fold (Kramer et al., 1992; Brummell and Labavitch, 1997), suggesting that the observed increases in gross viscosity were due to an increase in the viscosity of the soluble components of the juice. Similarly, in transgenic lines suppressed in PME activity, serum viscosity, whole juice viscosity, paste viscosity and serum separation were all improved relative to controls (Thakur et al., 1996a). Pectins from PME-suppressed lines had a much higher degree of methoxylation than controls, and since pectins that have not been demethylesterified by PME are inaccessible to PG (Tieman et al., 1992), they were protected from depolymerization during juice preparation (Thakur et al., 1996b). This resulted in an increase in serum viscosity of up to 3.5-fold in PME-suppressed lines, depending on processing conditions (Thakur et al., 1996a). In LeExp1-suppressed fruit, paste gross viscosity was substantially increased (by almost 20%) although serum viscosity was not improved relative to controls (Table 2). This suggests that the enhancement in viscosity of paste was due to a change in the properties of the insoluble, particulate components, or on the interaction of the
insoluble material with the soluble serum. However, it should be cautioned that the method used to prepare paste will over-estimate the contribution of the particulates to paste viscosity by increasing interparticle interaction, which affects viscosity (Beresovsky et al., 1995). The increase in wall firmness due to suppression of LeExp1 activity may affect the shape, size or configuration of cell wall fragments, including cellulose microfibrils, in the juice. The form and amount of cell wall fragments, which are approximately half of the insoluble solids, are a very important contributor to rheological properties and are essential for high viscosity in juice or paste (Whittenberger and Nutting, 1957, 1958; Tanglertpaibul and Rao, 1987). It would be very interesting to cross together LeExp1- and PGsuppressed transgenic lines and examine juice and paste prepared from fruit deficient in both activities. The increased viscosity of the particulates caused by suppression of LeExp1 combined with the increased viscosity of the serum caused by suppression of PG might produce a paste with exceptional rheological properties.
Acknowledgements We thank Carol McGugin for assistance with pathogen assays, Minh Ho for fruit weight measurements, Jacquie de Silva and Montan˜ a Ca´ mara for advice on laboratory juice and paste preparation, and Kristen Natoli, Vinnie Pirozzi and Veronica Ochoa for care and maintenance of plants. This work was supported by Seminis Vegetable Seeds.
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